Nanoenzymes

Fe3O4NP@DAAO. 

NP-DAAO

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This system is composed of magnetic iron oxide NPs conjugated with the ROS generating enzyme D-aminoacid oxidase (DAAO). Fe3O4 NP@DAAO wants to combine the advantages of magnetic NPs (low-toxicity, specific target by a magnetic field, ability to cross biological barriers) with those of DAAO (no-toxicity, easy modulation of the activity, hence of ROS generation). The system can be i.v. Injected and addressed by an external magnetic field in the tumor area. Here D-amino acids (naturally present or externally injected) act as substrate for the enzyme, causing H2O2 production and tumor cell death by apoptosis.

In industrial enzymatic application processes, immobilization of enzymes can offer several advantages, including the ability to re-use repeatedly, improvement of enzyme stability and broadening the optimum pH range of enzyme.
In our laboratory, in collaboration with the interuniversity center “The protein Factory”, we are immobilizing enzymes using magnetic NPs. The work is in progress for the systems:

    • NP@ACYLASE. This enzymatic approach is a one-step process in which CephC is directly converted to 7-ACA by a true CephC acylase. This approach is very attractive at the industrial level because of the prospects of simplifying the process and reducing costs.
    • VAC@Acylase. With the aim to further improve the one-step conversion of CephC to 7-ACA H296S-H309S VAC acylase was immobilized to Fe3O4 magnetic NPs. This conjugation could allow a simplest reuse and a greater stability of the enzyme, leading to a reduction of costs.
    • NP@LASPO. The last enzyme system for industrial application designed in this study is composed of Fe3O4@APTES NPs conjugated to L-aspartate oxidase (LASPO). LASPO is a prokaryotic enzyme that catalyzes the first step of de novo nicotinamide adenine dinucleotide (NAD+) biosynthesis. Recombinant StLASPO was efficiently used for the resolution of a racemic mixture of D, L-aspartate: low amount of StLASPO (9U) allowed to reach quantitative conversion and >99.5% e.e. Indeed and because of the high stability, this enzyme can be used for additional cycles of bioconversion. Conjugation on a support like NPs could further improve StLASPO qualities.

Methods.

    1. Evolution of new NPs-enzyme systems for biotechnological applications.
    2. Evaluation of cell toxicity and distribution.
    3. Evaluation of tissue distribution.
    4. Enzymology: steady state and pre-steady state analyses.

Department of biotechnology and Life Science – DBSV